Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Altered kidney distribution and loss of ACE2 into the urine in acute kidney injury.

There are diverse pathophysiological mechanisms involved in acute kidney injury (AKI). Among them, overactivity of the renin-angiotensin system (RAS) has been described. Angiotensin-converting enzyme 2 (ACE2) is a tissue RAS enzyme expressed in the apical border of proximal tubules. Given the important role of ACE2 in the metabolism of angiotensin II, this study aimed to characterize kidney and urinary ACE2 in a mouse model of AKI. Ischemia-reperfusion injury (IRI) was induced in C57BL/6 mice by clamping of the left renal artery followed by removal of the right kidney. In kidneys harvested 48 h after IRI, immunostaining revealed a striking maldistribution of ACE2 including spillage into the tubular lumen and the presence of ACE2-positive luminal casts in the medulla. In cortical membranes, ACE2 protein and enzymatic activity were both markedly reduced (37 ± 4 vs. 100 ± 6 ACE2/ß-actin, P = 0.0004, and 96 ± 14 vs. 152 ± 6 RFU/µg protein/h, P = 0.006). In urine, full-length membrane-bound ACE2 protein (100 kDa) was markedly increased (1,120 ± 405 vs. 100 ± 46 ACE2/µg creatinine, P = 0.04), and casts stained for ACE2 were recovered in the urine sediment. In conclusion, in AKI caused by IRI, there is a marked loss of ACE2 from the apical tubular border with deposition of ACE2-positive material in the medulla and increased urinary excretion of full-length membrane-bound ACE2 protein. The deficiency of tubular ACE2 in AKI suggests that provision of this enzyme could have therapeutic applications and that its excretion in the urine may also serve as a diagnostic marker of severe proximal tubular injury.NEW & NOTEWORTHY This study provides novel insights into the distribution of kidney ACE2 in a model of AKI by IRI showing a striking detachment of apical ACE2 from proximal tubules and its loss in urine and urine sediment. The observed deficiency of kidney ACE2 protein and enzymatic activity in severe AKI suggests that administration of forms of this enzyme may mitigate AKI and that urinary ACE2 may serve as a potential biomarker for tubular injury.

Histone deacetylase expression following cisplatin-induced acute kidney injury in male and female mice.

The chemotherapeutic agent cisplatin accumulates in the kidneys, leading to acute kidney injury (AKI). Preclinical and clinical studies have demonstrated sex-dependent outcomes of cisplatin-AKI. Deranged histone deacetylase (HDAC) activity is hypothesized to promote the pathogenesis of male murine cisplatin-AKI; however, it is unknown whether there are sex differences in the kidney HDACs. We hypothesized that there would be sex-specific Hdac expression, localization, or enzymatic activity, which may explain sexual dimorphic responses to cisplatin-AKI. In normal human kidney RNA samples, HDAC10 was significantly greater in the kidneys of women compared with men, whereas HDAC1, HDAC6, HDAC10, and HDAC11 were differentially expressed between the kidney cortex and medulla, regardless of sex. In a murine model of cisplatin-AKI (3 days after a 15 mg/kg injection), we found few sex- or cisplatin-related differences in Hdac kidney transcripts among the mice. Although Hdac9 was significantly greater in female mice compared with male mice, HDAC9 protein localization did not differ. Hdac7 transcripts were greater in the inner medulla of cisplatin-AKI mice, regardless of sex, and this agreed with a greater HDAC7 abundance. HDAC activity within the cortex, outer medulla, and inner medulla was significantly lower in cisplatin-AKI mice but did not differ between the sexes. In agreement with these findings, a class I HDAC inhibitor did not improve kidney injury or function. In conclusion, even though cisplatin-AKI was evident and there were transcript level differences among the different kidney regions in this model, there were few sex- or cisplatin-dependent effects on kidney HDAC localization or activity.NEW & NOTEWORTHY Kidney histone deacetylases (HDACs) are abundant in male and female mice, and the inner medulla has the greatest HDAC activity. A low dose of cisplatin caused acute kidney injury (AKI) in these mice, but there were few changes in kidney HDACs at the RNA/protein/activity level. A class I HDAC inhibitor failed to improve AKI outcomes. Defining the HDAC isoform, cellular source, and interventional timing is necessary to determine whether HDAC inhibition is a therapeutic strategy to prevent cisplatin-AKI in both sexes.

SARS-CoV-2 Spike Protein Enhances Carboxypeptidase Activity of Angiotensin-Converting Enzyme 2.

In this study, we investigated whether severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein may modify angiotensin-converting enzyme 2 (ACE2) activity in the plasma, heart, kidney, liver, lung, and six brain regions (amygdala, brain stem, cortex, hippocampus, hypothalamus, and striatum) of diabetic and hypertensive rats. We determine ACE2 activity in the plasma and lysates of heart, kidney, liver, lung, and six brain regions. MLN-4760 inhibits ACE2 activity in the plasma and all organs. On the other hand, soluble ACE2 (sACE2) activity increased in the plasma of diabetic rats, and there was no change in the plasma of hypertensive rats. ACE2 activity was augmented in the liver, brain stem, and striatum, while it decreased in the kidney, amygdala, cortex, and hippocampus of diabetic rats. ACE2 activity increased in the kidney, liver, and lung, while it decreased in the heart, amygdala, cortex, and hypothalamus of hypertensive rats. We measured the ACE2 content via enzyme-linked immunosorbent assay and found that ACE2 protein levels increased in the heart, while it decreased in the plasma, kidney, brain stem, cortex, hippocampus, hypothalamus, and striatum of diabetic rats. ACE2 protein levels decreased in the brain stem, cortex, hippocampus, and hypothalamus of hypertensive rats. Our data showed that the spike protein enhanced ACE2 activity in the liver and lungs of diabetic rats, as well as in the heart and three of the brain regions (cortex, hypothalamus, and striatum) of hypertensive rats.

The Na+,K+-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state.

The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2-BeFx). E2-BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9-7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2-BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.

Targeting lysine-specific demethylase 1A inhibits renal epithelial-mesenchymal transition and attenuates renal fibrosis.

Lysine-specific histone demethylase 1 (LSD1) as the first identified histone/lysine demethylase regulates gene expression and protein functions in diverse diseases. In this study, we show that the expression of LSD1 is increased in mouse kidneys with unilateral ureteral obstruction (UUO) and in cultured NRK-52E cells undergoing TGF-ß1-induced epithelial-mesenchymal transition (EMT). Inhibition of LSD1 with its specific inhibitor ORY1001 attenuated renal EMT and fibrosis, which was associated with decreased the deposition of extracellular matrix proteins and the expression of fibrotic markers, including α-smooth muscle actin (α-SMA) and fibronectin, and the recovery of E-cadherin expression and decrease of N-cadherin expression in UUO kidneys and in NRK-52E cells induced with TGF-ß1. Targeting LSD1 also decreased the expression of Snail family transcriptional repressor 1 (Snail-1) and its interaction with LSD1 in UUO kidneys and in NRK-52E cells treated with TGF-ß1. In addition, we identified a novel LSD1-14-3-3ζ-PKCα axis in the regulation of the activation of AKT and Stat3 and then the activation of fibroblasts. This study suggests that LSD1 plays a critical role in regulation of renal EMT and fibrosis through activation of diverse signaling pathways and places an emphasis that LSD1 has potential as a therapeutic target for the treatment of renal fibrosis.

Antioxidant enzyme peroxiredoxin 5 regulates cyst growth and ciliogenesis via modulating Plk1 stability.

Oxidative stress is emerging as a contributing factor to the homeostasis in cystic diseases. However, the role antioxidant enzymes play in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) remains elusive. Peroxiredoxin 5 (Prdx5) is an antioxidant enzyme that catalyzes the reduction of H2 O2 and alkyl hydroperoxide and plays an important role in different biological processes. In this study, we show that Prdx5 is downregulated in a PKD mutant mouse model and ADPKD patient kidneys. Knockdown of Prdx5 resulted in the formation of cysts in a three-dimensional mouse inner medullar collecting duct (IMCD) cell Matrigel culture system. The mechanisms of Prdx5 deficiency mediated cyst growth include: (1) induction of oxidative stress as indicated by increased mRNA expression of heme oxygenase-1, an oxidant stress marker; (2) activation of Erk, S6 and mTORC1, which contribute to cystic renal epithelial cell proliferation and cyst growth; (3) abnormal centrosome amplification and multipolar spindle formation which result in genome instability; (4) upregulation of Polo-like kinase 1 (Plk1) and Aurora kinase A, important mitotic kinases involved in cell proliferation and ciliogenesis; (5) impaired formation of primary cilia in mouse IMCD3 and retinal pigment epithelial cells, which could be rescued by inhibiting Plk1 activity; and (6) restraining the effect of Wnt3a and Wnt5a ligands on primary cilia in mouse IMCD3 cells, while regulating the activity of the canonical and non-canonical Wnt signaling in a separate cilia independent mechanism, respectively. Importantly, we found that targeting Plk1 with its inhibitor, volasertib, delayed cyst growth in Pkd1 conditional knockout mouse kidneys. Together, these findings indicate that Prdx5 is an important antioxidant that regulates cyst growth via diverse mechanisms, in particular, the Prdx5-Plk1 axis, and that induction and activation of Prdx5, alone or together with inhibition of Plk1, represent a promising strategy for combatting ADPKD.

Substituted kynurenic acid derivatives as fluorophore-based probes for D- and L-amino acid oxidase assays and their in vitro application in eels.

High levels of D-amino acid oxidase (DAO) are associated with neurological and psychiatric disorders, while L-amino acid oxidase (LAO) exhibits antimicrobial and antitumor properties. The enzymatic conversion of the non-fluorescent kynurenine (KYN) into the endogenous weak fluorescent kynurenic acid (KYNA) by the action of DAO has previously been reported. However, the fluorescence of KYNA can be improved by changing the substituents on the aromatic rings. In this study, we prepared different 6-phenyl-substituted KYNA derivatives and investigated their fluorescence properties. Among them, 2-MePh-KYNA showed the maximum fluorescence quantum yield of 0.881 at 340 nm excitation and 418 nm emission wavelengths. The effects of solvent properties (dielectric constant, pKa, viscosity, and proticity) on the fluorescence intensity (FLI) of the KYNA derivatives were explored. The FLI of 2-MePh-KYNA was significantly large in protic solvents. Subsequently, 2-MePh-D-KYN and 2-MePh-L-KYN were prepared with high enantiopurity (>99.25%) for the enzymatic conversion. 2-MePh-D-KYN exhibited high sensitivity (∼19 times that of a commercial DAO substrate and ∼60 times that of the previously reported MeS-D-KYN) and high selectivity, as it was not cross-reactive towards LAO, while 2-MePh-L-KYN was also converted into 2-MePh-KYNA by LAO. Furthermore, the 2-MePh-D-KYN probe successfully detected DAO in eel liver, kidney, and heparin-anticoagulated plasma in the in vitro study.

XPF-ERCC1 protects liver, kidney and blood homeostasis outside the canonical excision repair pathways.

Loss of the XPF-ERCC1 endonuclease causes a dramatic phenotype that results in progeroid features associated with liver, kidney and bone marrow dysfunction. As this nuclease is involved in multiple DNA repair transactions, it is plausible that this severe phenotype results from the simultaneous inactivation of both branches of nucleotide excision repair (GG- and TC-NER) and Fanconi anaemia (FA) inter-strand crosslink (ICL) repair. Here we use genetics in human cells and mice to investigate the interaction between the canonical NER and ICL repair pathways and, subsequently, how their joint inactivation phenotypically overlaps with XPF-ERCC1 deficiency. We find that cells lacking TC-NER are sensitive to crosslinking agents and that there is a genetic interaction between NER and FA in the repair of certain endogenous crosslinking agents. However, joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice. These analyses indicate that XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage. Failure to resolve such damage leads to loss of tissue homeostasis in mice and humans.

Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility.

Aged males disproportionately succumb to increased COVID-19 severity, hospitalization, and mortality compared to females. Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) facilitate SARS-CoV-2 viral entry and may have sexually dimorphic regulation. As viral load dictates disease severity, we investigated the expression, protein levels, and activity of ACE2 and TMPRSS2. Our data reveal that aged males have elevated ACE2 in both mice and humans across organs. We report the first comparative study comprehensively investigating the impact of sex and age in murine and human levels of ACE2 and TMPRSS2, to begin to elucidate the sex bias in COVID-19 severity.

15-Lipoxygenase worsens renal fibrosis, inflammation, and metabolism in a murine model of ureteral obstruction.

15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.

Role of heme oxygenase in the regulation of the renal hemodynamics in a model of sex-dependent programmed hypertension by maternal diabetes.

Intrauterine programming of cardiovascular and renal function occurs in diabetes because of the adverse maternal environment. Heme oxygenase 1 (HO-1) and -2 (HO-2) exert vasodilatory and antioxidant actions, particularly in conditions of elevated HO-1 expression or deficient nitric oxide levels. We evaluated whether the activity of the heme-HO system is differentially regulated by oxidative stress in the female offspring of diabetic mothers, contributing to the improved cardiovascular function in comparison with males. Diabetes was induced in pregnant rats by a single dose of streptozotocin (STZ, 50 mg/kg ip) in late gestation. Three-month-old male offspring from diabetic mothers (MODs) exhibited higher blood pressure (BP), higher renal vascular resistance (RVR), worse endothelium-dependent response to acetylcholine (ACH), and an increased constrictor response to phenylephrine (PHE) compared with those in age-matched female offspring of diabetic mothers (FODs), which were abolished by chronic tempol (1 mM) treatment. In anesthetized animals, stannous mesoporphyrin (SnMP; 40 µmol/kg iv) administration, to inhibit HO activity, increased RVR in FODs and reduced glomerular filtration rate (GFR) in MODs, without altering these parameters in control animals. When compared with MODs, FODs showed lower nitrotirosyne levels and higher HO-1 protein expression in renal homogenates. Indeed, chronic treatment with tempol in MODs prevented elevations in nitrotyrosine levels and the acute renal hemodynamics response to SnMP. Then, maternal diabetes results in sex-specific hypertension and renal alterations associated with oxidative stress mainly in adult male offspring, which are reduced in the female offspring by elevation in HO-1 expression and lower oxidative stress levels.

Critical Role of Neprilysin in Kidney Angiotensin Metabolism.

RATIONALE: Kidney homeostasis is critically determined by the coordinated activity of the renin-angiotensin system (RAS), including the balanced synthesis of its main effector peptides Ang (angiotensin) II and Ang (1-7). The condition of enzymatic overproduction of Ang II relative to Ang (1-7) is termed RAS dysregulation and leads to cellular signals, which promote hypertension and organ damage, and ultimately progressive kidney failure. ACE2 (angiotensin-converting enzyme 2) and NEP (neprilysin) induce the alternative, and potentially reno-protective axis by enhancing Ang (1-7) production. However, their individual contribution to baseline RAS balance and whether their activities change in chronic kidney disease (CKD) has not yet been elucidated. OBJECTIVE: To examine whether NEP-mediated Ang (1-7) generation exceeds Ang II formation in the healthy kidney compared with diseased kidney. METHODS AND RESULTS: In this exploratory study, we used liquid chromatography-tandem mass spectrometry to measure Ang II and Ang (1-7) synthesis rates of ACE, chymase and NEP, ACE2, PEP (prolyl-endopeptidase), PCP (prolyl-carboxypeptidase) in kidney biopsy homogenates in 11 healthy living kidney donors, and 12 patients with CKD. The spatial expression of RAS enzymes was determined by immunohistochemistry. Healthy kidneys showed higher NEP-mediated Ang (1-7) synthesis than Ang II formation, thus displaying a strong preference towards the reno-protective alternative RAS axis. In contrast, in CKD kidneys higher levels of Ang II were recorded, which originated from mast cell chymase activity. CONCLUSIONS: Ang (1-7) is the dominant RAS peptide in healthy human kidneys with NEP rather than ACE2 being essential for its generation. Severe RAS dysregulation is present in CKD dictated by high chymase-mediated Ang II formation. Kidney RAS enzyme analysis might lead to novel therapeutic approaches for CKD.

Glutathionylation Decreases Methyltransferase Activity of PRMT5 and Inhibits Cell Proliferation.

Glutathionylation is an important posttranslational modification that protects proteins from further oxidative damage as well as influencing protein structure and activity. In the present study, we demonstrate that the cysteine-42 residue in protein arginine N-methyltransferase 5 (PRMT5) is glutathionylated in aged mice or in cells that have been exposed to oxidative stress. Deglutathionylation of this protein is catalyzed by glutaredoxin-1 (Grx1). Using mutagenesis and subsequent biochemical analyses, we show that glutathionylation decreased the binding affinity of PRMT5 with methylosome protein-50 (MEP50) and reduced the methyltransferase activity of PRMT5. Furthermore, overexpression of PRMT5-C42A mutant caused a significant increase in histone methylation in HEK293T and A549 cells and promoted cell growth, whereas overexpression of the PRMT5-C42D mutant, a mimic of glutathionylated PRMT5, inhibited cell proliferation. Taken together, our results demonstrate a new mechanism of regulation of PRMT5 methyltransferases activity and suggest that PRMT5 glutathionylation is partly responsible for reactive oxygen species-mediated cell growth inhibition.

[Intervention effects of drugs on GSH and SOD enzyme activity of rats kidney acutely poisoned by nickel carbonyl].

Objective: To evaluate the intervention effect of various drugs on glutathione (GSH) and superoxide dismutase (SOD) enzyme activity of rats kidney with acute nickel carbonyl poisoning. Methods: In January 2019, The 250 SPF male SD rats were randomly divided into normal control group (n=10) , poisoned group (n=40) and treatment groups (n=200) according to the random number table method. And the treatment groups were divided into methylprednisolone group (20 mg/kg) , DDC group (100 mg/kg) , sodium selenite group (10 µmol/kg) , Shenfu huiyang decoction group (0.25 ml) and methylprednisolone combined with DDC group (100 mg/kg) , with 40 mice in each group. Except for the normal control group, rats in the other groups were exposed to nickel carbonyl for 30 min, at 4 h and 30 h after exposure, the rats in each treatment group were intraperitoneally injected with corresponding drugs, and kidney tissues were collected 3 d and 7 d after administration, with 10 mice in each group. The activities of GSH and SOD in kidney were measured by enzyme-linked immunosorbent assay, and using electron microscopy observe ultrastructure changes. Results: Compared to the control group, the activities of GSH and SOD enzyme of poisoned group were significantly decreased at 3 d or 7 d after 4 h or 30 h exposure, and the difference was statistically significant (P=0.000, 0.031, 0.001, 0.033) , the epithelial nuclei of proximal convoluted tubules were pyknosis and lysosome hyperplasia in the cytoplasm. And compared to poisoned group, the activities of GSH and SOD enzyme of methylprednisolone+DDC group were significantly increased at treatment with 7 d after 4 h exposure, the difference was statistically significant (P=0.022, 0.000) , and the activities of GSH and SOD enzyme of methylprednisolone and enzyme of methylprednisolone+DDC group were significantly higher at 7 days than at 3 days, the difference was statistically significant (P=0.020, 0.017, 0.018, 0.033) . The results of electron microscopy showed that the cell nuclei and cytoplasmic organelles of proximal convolute tubule were almost restored to normal tissue level of both methylprednisolone group and methylprednisolone+DDC group. Conclusion: The methylprednisolone and methylprednisolone+DDC have obvious repair effect on renal enzyme activity level of rats with acute nickel carbonyl poisoning, and the treatment effect is better for a long time of medication.

Dipeptidyl peptidase 3 modulates the renin-angiotensin system in mice.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase involved in degrading oligopeptides with 4-12 amino acid residues. It has been associated with several pathophysiological processes, including blood pressure regulation, pain signaling, and cancer cell defense against oxidative stress. However, the physiological substrates and the cellular pathways that are potentially targeted by DPP3 to mediate these effects remain unknown. Here, we show that global DPP3 deficiency in mice (DPP3-/-) affects the renin-angiotensin system (RAS). LC-MS-based profiling of circulating angiotensin peptides revealed elevated levels of angiotensin II, III, IV, and 1-5 in DPP3-/- mice, whereas blood pressure, renin activity, and aldosterone levels remained unchanged. Activity assays using the purified enzyme confirmed that angiotensin peptides are substrates for DPP3. Aberrant angiotensin signaling was associated with substantially higher water intake and increased renal reactive oxygen species formation in the kidneys of DPP3-/- mice. The metabolic changes and altered angiotensin levels observed in male DPP3-/- mice were either absent or attenuated in female DPP3-/- mice, indicating sex-specific differences. Taken together, our observations suggest that DPP3 regulates the RAS pathway and water homeostasis by degrading circulating angiotensin peptides.

The ERK1/2-ATG13-FIP200 signaling cascade is required for autophagy induction to protect renal cells from hypoglycemia-induced cell death.

Autophagy, an evolutionarily conserved lysosomal degradation pathway, is known to regulate a variety of physiological and pathological processes. At present, the function and the precise mechanism of autophagy regulation in kidney and renal cells remain elusive. Here, we explored the role of ERK1 and ERK2 (referred as ERK1/2 hereafter) in autophagy regulation in renal cells in response to hypoglycemia. Glucose starvation potently and transiently activated ERK1/2 in renal cells, and this was concomitant with an increase in autophagic flux. Perturbing ERK1/2 activation by treatment with inhibitors of RAF or MEK1/2, via the expression of a dominant-negative mutant form of MEK1/2 or RAS, blocked hypoglycemia-mediated ERK1/2 activation and autophagy induction in renal cells. Glucose starvation also induced the accumulation of reactive oxygen species in renal cells, which was involved in the activation of the ERK1/2 cascade and the induction of autophagy in renal cells. Interestingly, ATG13 and FIP200, the members of the ULK1 complex, contain the ERK consensus phosphorylation sites, and glucose starvation induced an association between ATG13 or FIP200 and ERK1/2. Moreover, the expression of the phospho-defective mutants of ATG13 and FIP200 in renal cells blocked glucose starvation-induced autophagy and rendered cells more susceptible to hypoglycemia-induced cell death. However, the expression of the phospho-mimic mutants of ATG13 and FIP200 induced autophagy and protected renal cells from hypoglycemia-induced cell death. Taken together, our results demonstrate that hypoglycemia activates the ERK1/2 signaling to regulate ATG13 and FIP200, thereby stimulating autophagy to protect the renal cells from hypoglycemia-induced cell death.

Expression and function of human ribonuclease 4 in the kidney and urinary tract.

Antimicrobial peptides are essential host defense mechanisms that prevent urinary tract infections. Recent studies have demonstrated that peptides in the ribonuclease A superfamily have antimicrobial activity against uropathogens and protect the urinary tract from uropathogenic Escherichia coli (UPEC). Little is known about the antibacterial function or expression of ribonuclease 4 (RNase 4) in the human urinary tract. Here, we show that full-length recombinant RNase 4 peptide and synthetic amino-terminal RNase 4 peptide fragment have antibacterial activity against UPEC and multidrug-resistant (MDR)-UPEC. RNASE4 transcript expression was detected in human kidney and bladder tissue using quantitative real-time PCR. Immunostaining or in situ hybridization localized RNase 4 expression to proximal tubules, principal and intercalated cells in the kidney's collecting duct, and the bladder urothelium. Urinary RNase 4 concentrations were quantified in healthy controls and females with a history of urinary tract infection. Compared with controls, urinary RNase 4 concentrations were significantly lower in females with a history of urinary tract infection. When RNase 4 was neutralized in human urine or silenced in vitro using siRNA, urinary UPEC replication or attachment to and invasion of urothelial and kidney medullary cells increased. These data show that RNase 4 has antibacterial activity against UPEC, is expressed in the human urinary tract, and can contribute to host defense against urinary tract infections.NEW & NOTEWORTHY Ribonuclease 4 (RNase 4) is a newly identified host defense peptide in the human kidney and bladder. RNase 4 kills uropathogenic Escherichia coli (UPEC) and multidrug-resistant UPEC. RNase 4 prevents invasive UPEC infection and suppressed RNase 4 expression may be a risk factor for more severe or recurrent urinary tract infection.

Semaporin3A inhibitor ameliorates renal fibrosis through the regulation of JNK signaling.

Renal fibrosis is the common pathological pathway in progressive renal diseases. In the present study, we analyzed the roles of semaphorin 3 A (SEMA3A) on renal fibrosis and the effect of SEMA3A inhibitor (SEMA3A-I) using a unilateral ureteral obstruction (UUO) mouse model. Expression of SEMA3A in the proximal tubulus and neuropilin-1, a recepor of SEMA3A, in fibloblast and tubular cells were increased in UUO kidneys. The expression of myofibroblast marker tenascin-C and fibronection as well as renal fibrosis were increased in UUO kidneys, all of which were ameliorated by SEMA3A-I. In addition, the JNK signaling pathway, known as the target of SEMA3A signaling, was activated in proximal tubular cells and fibroblast cells after UUO surgery, and SEMA3A-I significantly attenuated the activation. In vitro, treatments with SEMA3A as well as transforming growth factor-ß1 (TGF-ß1) in human proximal tubular cells lost epithelial cell characteristics, and SEMA3A-I significantly ameliorated this transformation. The JNK inhibitor SP600125 partially reversed SEMA3A and TGF-ß1-induced cell transformation, indicating that JNK signaling is involved in SEMA3A-induced renal fibrosis. In addition, treatment with SEMA3A in fibroblast cells activated expression of tenascin-C, collagen type I, and fibronection, indicating that SEMA3A may accelerate renal fibrosis through the activation of fibroblast cells. Analysis of human data revealed the positive correlation between urinary SEMA3A and urinary N-acetyl-ß-d-glucosaminidase, indicating the association between SEMA3A and tubular injury. In conclusion, SEMA3A signaling is involved in renal fibrosis through the JNK signaling pathway and SEMA3A-I might be a therapeutic option for protecting from renal fibrosis.NEW & NOTEWORTHY Renal fibrosis is the common pathological pathway in the progression of renal diseases. This study, using a unilateral ureteral obstruction (UUO) mouse model, indicated increased semaphorin3A (SEMA3A) signaling in renal tubular cells as well as fibroblast cells under UUO surgery, and SEMA3A inhibitor ameliorated UUO-induced renal fibrosis through the regulation of JNK signaling. The study proposes the potential therapeutic option of SEMA3A inhibitor to treat renal fibrosis.

Expression of lactate dehydrogenase A and B isoforms in the mouse kidney.

Cellular metabolic rates in the kidney are critical for maintaining normal renal function. In a hypoxic milieu, cells rely on glycolysis to meet energy needs, resulting in the generation of pyruvate and NADH. In the absence of oxidative phosphorylation, the continuation of glycolysis is dependent on the regeneration of NAD+ from NADH accompanied by the fermentation of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase (LDH) isoform A (LDHA), whereas LDH isoform B (LDHB) catalyzes the opposite reaction. LDH is widely used as a potential injury marker as it is released from damaged cells into the urine and serum; however, the precise isoform-specific cellular localization of the enzyme along the nephron has not been characterized. By combining immunohistochemistry results and single-cell RNA-sequencing data on healthy mouse kidneys, we identified that LDHA is primarily expressed in proximal segments, whereas LDHB is expressed in the distal parts of the nephron. In vitro experiments in mouse and human renal proximal tubule cells showed an increase in LDHA following hypoxia with no change in LDHB. Using immunofluorescence, we observed that the overall expression of both LDHA and LDHB proteins decreased following renal ischemia-reperfusion injury as well as in the adenine-diet-induced model of chronic kidney disease. Single-nucleus RNA-sequencing analyses of kidneys following ischemia-reperfusion injury revealed a significant decline in the number of cells expressing detectable levels of Ldha and Ldhb; however, cells that were positive showed increased average expression postinjury, which subsided during the recovery phase. These data provide information on the cell-specific expression of LDHA and LDHB in the normal kidney as well as following acute and chronic kidney disease.NEW & NOTEWORTHY Cellular release of lactate dehydrogenase (LDH) is being used as an injury marker; however, the exact localization of LDH within the nephron remains unclear. We show that LDH isoform A is expressed proximally, whereas isoform B is expressed distally. Both subunit expressions were significantly altered in models of acute kidney injury and chronic kidney disease. Our study provides new insights into basal and postinjury renal lactate metabolism.